Abstract: As non-coding RNAs (ncRNA) are increasingly implicated in cellular functions as well as disease states, there is a need to deepen our understanding of RNA structure-function relationships as well as develop methods to target RNA with small molecules. The conventional view that ncRNAs fold into a single well-defined secondary structure was recently challenged by NMR relaxation dispersion experiments showing that non- canonical RNA motifs exist in dynamic equilibrium with alternative higher energy secondary structures referred to as `excited states' (ES). Mutagenesis studies suggest that RNA ESs exist for the vast majority of RNA molecules. The significantly altered structures of RNA ESs are correlated with altered biological activities potentially providing the basis for new regulatory RNA-based switches. In addition, because ESs trade canonical Watson-Crick base pairs for non-canonical mispairs, their structures are unique and complex potentially offering new receptors for the discovery of small molecules that bind RNA with high affinity and selectivity. Trapping inactive ES conformations of RNA with small molecules may lead to a new strategy for RNA-directed drug discovery. However, RNA ESs pose unique challenges to conventional structural and functional characterization techniques because they exist transiently in low abundance. We propose to develop a new paradigm to structurally and functionally characterize RNA ESs as well as to test their suitability as drug targets with specific application to one of two ESs that have recently been uncovered in human immunodeficiency virus type-1 (HIV-1) transactivation response element (TAR) RNA. Aim 1 will validate the TAR ES as a therapeutic target by stabilizing the ES conformation using a mutagenesis approach and testing the hypothesis that mutations that trap the ES significantly alter TAR's activity in HIV-1 transcription. Aim 2 will solve a dynamic structural ensemble of the TAR ES using a new strategy that combines X-ray crystallography, NMR and Molecular Dynamics Simulations. Aim 3 will use a structure-guided approach to identify novel classes of anti-HIV small molecules that bind and stabilize the TAR ES. This work will help evaluate RNA ESs as new drug targets and provide a foundation for structural and functional characterization of other RNA ESs.